CN115645439A - Application of cell preservation solution in preparation of immunosuppressant - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to application of a cell preservation solution in preparation of an immunosuppressant. The invention provides application of a cell preservation solution in preparation of an immunosuppressant, and the cell preservation solution obtained by a specific method is applied to a preparation (such as an injection solution) containing mesenchymal stem cells, can endow the injection solution with a certain immunosuppressive function, can be combined with MSCs cells to improve the treatment effect, and can improve the long-time transport capacity and the survival rate of the MSCs.

Description

Application of cell preservation solution in preparation of immunosuppressant

Technical Field

The invention relates to the technical field of biology, in particular to application of a cell preservation solution in preparation of an immunosuppressant.

Background

Mesenchymal Stem Cells (MSCs) are pluripotent stem cells derived from mesoderm and have various functions, and among them, most of them, they have been studied for their immunoregulatory ability, and have been used for treating crohn's disease, GVHD, and the like in applications.

The physiological saline is the most common cell resuspension liquid, is the most common liquid for preserving liquid in the cell transportation process, belongs to clinical products, and has the advantages that the cell resuspension prepared in a laboratory is not required to be processed again, and can be directly used for clinic. The normal saline can be used in short-term transportation, which ensures the activity time of cells to be about 10 hours, but the activity of the cells gradually slides down along with the time in the long-term transportation process, so that long-distance transportation cannot be carried out, especially under the condition of preparing high-concentration injection.

There is a need to develop a preservation solution that can be used for long-term transport of MSC preparations.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, an object of the present invention is to provide a use of a cell preservation solution for preparing an immunosuppressant, which can impart a certain immunosuppressive function to an injection solution, improve a therapeutic effect by binding to MSCs cells, and improve long-term trafficability and survival rate of MSCs, by applying the cell preservation solution obtained by a specific method to a preparation containing mesenchymal stem cells (e.g., an injection solution).

The cell balls are formed through 3D and then cultured in physiological saline under certain conditions, the cell balls can secrete a large number of secretion factors which are favorable for cell survival under the conditions, the cells are removed after being cultured for certain time, only secretion products of the cells are reserved, and the culture supernatant is used in the cell transportation process of the MSCs cells, so that the cell protection in the transportation process is favorable. Can prolong the preservation time and improve the cell survival rate. And simultaneously, the immunosuppressive function of the MSCs is improved.

To this end, the present invention provides in a first aspect the use of a cell preservation solution in the preparation of an immunosuppressant. According to an embodiment of the present invention, the cell preservation solution is prepared by the following method:

(1) Preparing a 3D cell ball;

(2) Resuspending the 3D cell spheres and incubating to obtain a cell sphere suspension;

(3) Removing the cell fraction from the cell pellet suspension to obtain a cell preservation solution,

wherein the incubation temperature is 0-37 ℃, and the incubation time is 2-170 h.

A cytoprotective solution is a liquid specifically formulated for cell preservation, in which the activity of the cells is preserved for a relatively long period of time. So that the cells can maintain the activity of the cells in the interval of 0-20 ℃ for a longer time. However, the components of the heavy suspension liquid are often composed of non-clinical medicines, the heavy suspension liquid is often required to be removed again before use after long-distance transportation, on one hand, the use burden is increased, and the use cost is also increased, and the concentration of the conventional cell preservation liquid is often 1-2 x 10^6cells/mL, so that the conventional cell preservation liquid cannot be used for high-concentration injection.

The cell balls are formed through 3D and then cultured in physiological saline under certain conditions, the cell balls can secrete a large number of secretion factors which are favorable for cell survival and immunosuppression under the conditions, the cells are removed after being cultured for certain time, only secretion products of the cells are reserved, and the culture supernatant is used in the conventional cell transportation process, so that the cell transportation process is favorable for protecting the cells in the transportation process, and meanwhile, the cell transportation process has certain immunosuppression capacity. Can prolong the preservation time and improve the cell survival rate. The preservation solution can maintain the cell survival rate of more than 80% in 72h, has immunosuppressive function, and is suitable for long-term transportation. Is favorable for clinical use of cells and improvement of treatment effect.

According to an embodiment of the invention, the cells contained in the 3D cell spheres are selected from mesenchymal cells.

According to an embodiment of the present invention, the cells contained in the 3D cell pellet are selected from at least one of fibroblasts, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells.

According to an embodiment of the present invention, the method of forming the 3D cell spheres includes self-sphere and carrier sphere.

According to an embodiment of the present invention, in the step (2), the resuspension solution used for resuspending the 3D cell beads is at least one selected from the group consisting of sodium chloride injection, compound sodium chloride injection, sodium lactate ringer's injection, and glucose injection.

The used cell suspension is clinical injection, the safety is reliable, no exogenous protective substance is added, the use difficulty of long-distance transportation is reduced, and the cells can be directly used after the long-distance transportation. The protection liquid used in the mesenchymal stem cell transportation process can be effectively stored in a short time, but the cell viability can be rapidly reduced under the long-time transportation condition, the use difficulty is increased, by using the suspension of the technical scheme, the cell quantity and the cell viability of the mesenchymal stem cells can be effectively prolonged in the suspension of the technical scheme, the use convenience is increased, the viability of the cells at normal temperature can be prolonged in the suspension prepared by the technical scheme, and meanwhile, the content of the inflammation-inhibiting factor with higher content in the suspension can improve the curative effect of the mesenchymal stem cells on inflammation occurrence diseases in use.

According to an embodiment of the present invention, a method of preparing a 3D cell pellet includes:

1) Taking out the frozen cells for redissolution and resuspension;

2) Culturing the resuspended cells in DMEM medium containing 4-8% FBS;

3) Subjecting the cultured cells to trypsinization to obtain digested cells;

4) Resuspending the digested cells to obtain a cell resuspension solution;

5) And inoculating the cell re-suspension into a culture dish, and culturing to obtain the 3D cell ball.

According to an embodiment of the invention, the cells contained in the 3D cell spheres are selected from mesenchymal cells.

According to an embodiment of the present invention, the cells contained in the 3D cell pellet are selected from at least one of fibroblasts, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells.

According to an embodiment of the present invention, in step 4), the digested cells are resuspended using physiological saline.

According to an embodiment of the present invention, the immunosuppressive agent comprises mesenchymal stem cells.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows a flow diagram for preparing a cell pellet suspension according to one embodiment of the present invention;

FIG. 2A shows the effect of drug-treated MSCs on the proliferation-suppressing ability of immune cells in example 1 of the present invention; b is a graph showing the survival rate of cells after pretreatment of immune cells with the drug-treated suspension of example 1 of the present invention;

FIG. 3 shows the inhibition of immune cell proliferation by MSCs;

FIG. 4 shows the contents of TNF-. Beta.VEGF, hyaluronic acid, ANG-1 in MSCs 2D culture supernatants and 3D pellet culture supernatants;

FIG. 5 shows the proliferation inhibitory effect of culture supernatants on immune cells;

FIG. 6 shows the amount of secreted PGE-2 in different culture protocols;

FIG. 7 shows the proliferation inhibitory ability of the culture supernatants to immune cells;

FIG. 8 shows the cell morphology size after spheronization for different cell numbers;

FIG. 9 shows the passaged cell sphering state and the revived cell sphering state;

FIG. 10 shows group A in a 24 hour balled state 10X (Panel A); group B48 h cells were spheronized 20X (panel B); group C cells 72h sphered 20X (panel C);

FIG. 11 shows the group A cell pellet state and the group B cell pellet state;

FIG. 12 shows the effect of cell pellet incubation temperature on the survival status of cell pellets;

FIG. 13 shows the states of observation under a fluorescence microscope of a cell ball of groups A to C;

figure 14 demonstrates the investigation of the effect of different hatching fluid species on immune cell activity;

FIG. 15 shows the cell pellet morphology for groups A-D;

FIG. 16 shows the change in cell sphere diameter;

FIG. 17 shows the results of cell pellets in a normal flask;

FIG. 18 shows a phenotypic flow assay of MSCs;

fig. 19 shows results of adipogenic differentiation identification, chondrogenic differentiation identification, and osteogenic differentiation identification, respectively;

FIG. 20 shows a count plot of MSCs in suspension;

FIG. 21 shows an experiment of inhibition of immune cell proliferation by suspension;

FIG. 22 shows the toxicity test of cell pellet suspensions on hepatocytes;

FIG. 23 shows the morphology of cell spheres transferred to a low adsorption plate after two days of culture from 1w of cells prepared by the method of the present invention;

FIG. 24 shows the identification of intracellular morphology of cells within a cell sphere by staining of 1w cells prepared by the method of the present invention after incubation for 7 days;

FIG. 25 shows the adherence performance of cell balls placed in a wall plate prepared by the method of the embodiment of the invention;

FIG. 26 shows the cell status after 20h of staining of MSCs with Trypan blue.

Detailed Description

The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.

Reagents used in the experiments of examples are commercially available unless otherwise specified.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present disclosure, "a plurality" means at least two, e.g., two, three, etc., unless explicitly specifically limited otherwise.

The scheme of the present disclosure will be explained with reference to examples. It will be understood by those skilled in the art that the following examples are illustrative of the present disclosure only and should not be taken as limiting the scope of the present disclosure. The examples do not specify particular techniques or conditions, and are performed according to techniques or conditions described in literature in the art or according to the product specification. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 drug enhancement of cell sphere function

The MSC cells have certain immunoregulation capability, and the immunosuppressive capability of the MSCs is further improved by a medicament pretreatment mode.

The experimental process comprises the following steps: the MSCs cells in the flask were digested with 0.125% trypsin for 3 minutes, and the digestion was terminated with the culture supernatant. Washing primary cells with physiological saline, centrifuging to collect cells, diluting to a certain volume with physiological saline, counting, inoculating to a culture flask according to a density of 5000-7000 cells/cm 2, adding an appropriate amount of culture medium, culturing in an incubator containing 5% CO2 at 37 deg.C, sucking supernatant after 24 hours, adding new culture medium and screening drugs, culturing in the incubator for 24 hours, collecting supernatant, supplementing new culture medium without drugs, culturing for 24 hours again, and collecting supernatant.

Taking immune cells with high proliferation activity, centrifuging to remove culture medium, adjusting the density, inoculating into a culture plate, and adding 1 according to the following table 1:1 and culture medium. After 24 hours, CCK8 was added, and after incubation for 2 hours, the results were measured with a microplate reader.

TABLE 1

FIG. 2 is a graph A showing the effect of drug-treated MSCs on the proliferation-suppressing ability of immune cells, and a graph B showing the survival rate of cells after pretreatment of immune cells with drug-treated suspensions. And (4) conclusion: the MSC pretreated by the zoledronic acid has the advantages of improving the proliferation inhibition capability on immune cells, having no toxicity on the immune cells, and still having stronger immunosuppressive function under the condition that the zoledronic acid does not exist.

Example 2 comparison of 3D cell pellets with 2D cultured cells

The 3D structure formed by balling the MSC cells is beneficial to the cells to secrete factors related to higher cytoprotection, and simultaneously can increase the resistance of the MSCs to the environment and promote the survival of the cells, so that the 3D MSCs and the 2D cultured MSC cells are subjected to comparative test.

The experimental process comprises the following steps: the MSCs cells in the flask were digested with 0.125% pancreatin for 3 minutes, and the digestion was stopped with the culture supernatant. Washing cells with normal saline, centrifuging to collect cells, metering to a certain volume with normal saline, counting, taking out 500w MSC cells from two tubes, centrifuging again, removing supernatant, adding appropriate amount of fresh MSC culture medium, resuspending in a T75 bottle, adding cells from one tube to a 6cm plate cover by a suspension method, placing the cell suspension in a plate, and placing in 5% CO ₂ Culturing for 48 hours in an incubator, then taking out a culture flask and a flat dish, collecting all cell balls in the flat dish into a centrifuge tube, removing a supernatant culture solution, removing a culture supernatant in the culture flask, cleaning twice with physiological saline, then resuspending with ringer's solution, then placing in a refrigerator at 1-10 ℃ for 5 days, collecting the supernatant after 5 days, filtering with a filter, removing cells and cell debris, and collecting into a new centrifuge tube for later use in testing.

Table 2 shows the 3D cell pellet versus 2D culture comparative experimental design 1.

TABLE 2

Table 3 shows the 3D cell pellet versus 2D culture comparative experimental design 2.

TABLE 3

The co-culture of the cells, culture supernatant and immune cells was used to confirm whether the proliferation inhibitory effect on immune cells exerted a paracrine function or the cells themselves.

Table 4 shows the inhibition of immune cell proliferation by 3D cell balls and 2D cultures.

TABLE 4

Fig. 3 shows the inhibition of immune cell proliferation by MSCs, conclusion: the result of co-culture of the immune cells shows that the MSCs cultured by 3D have better inhibition capability on the immune cells, and the inhibition capability is obviously better than that of the MSCs cultured by 2D. Secondly, the inhibitory effect of the 3D culture supernatant was comparable to that of 3D beads themselves co-cultured with immune cells.

The comparison of the contents of TNF-. Beta.VEGF, hyaluronic acid, and ANG-1 in the MSCs 2D culture supernatants and the 3D pellet culture supernatants is shown in FIG. 4.

And (4) conclusion: the comparison of the contents of TNF-beta and VEGF in the MSCs 2D culture supernatant and the 3D sphere culture supernatant proves that the MSCs cultured in the 3D culture have higher contents of TNF-beta, VEGF and hyaluronic acid, but the ANG-1 has no obvious difference.

EXAMPLE 3 Effect of different culture regimes on the ability to suppress immune cells

The 3D cultured MSCs ball has strong inhibition capacity, and the 3D cell ball can survive in the ringer's solution for a long time and has certain protection capacity. Therefore, the supernatants obtained by incubating the cell balls with ringer's solution were compared with the culture medium supernatants to compare the suppression of the proliferation of the immune cells.

The experimental process comprises the following steps: preparing 3D culture medium supernatant; preparing a cell ball according to 2.3.2, transferring the prepared cell ball to a low adsorption plate, adding a proper amount of culture medium, placing the cell ball in an incubator for culture, collecting culture supernatant after culturing for 5 days, and filtering the culture supernatant by using a 0.22um filter for later use.

3D ringer supernatant; the supernatant of the 3D cell Glynerger's fluid was prepared according to 2.3.2 for use.

2D culture supernatant; inoculating 500w of cells into a new culture bottle, adding a proper amount of culture medium, culturing for 5 days, collecting supernatant, and filtering for later use.

Ringer's solution; ringer's solution without cell treatment.

A culture medium; a culture medium for culturing MSCs, wherein,

samples were prepared as described above. Taking immune cells with high proliferation activity, centrifuging to remove culture supernatant, adding new culture medium, adjusting to proper density, inoculating to a culture plate, and performing culture according to the following steps of 1:1 amount of the sample was added to the culture plate, and the cell viability and cell number of the immune cells were calculated by trypan blue at 48 hours, and the results are shown in table 5.

TABLE 5

Numbering	Average density	The activity ratio%
			3D Medium supernatant	8.39	91.7％
3D ringer supernatant	8.26	91.5％
			2D culture supernatant	9.1775	89.6％
Ringer's solution	11.2	90.0％
			Culture medium	12.4	94.0％

The proliferation inhibitory effect of the culture supernatant on immune cells is shown in FIG. 5.

Example 4 balling experiments with MSCs

There are various methods for 3D cell culture, and the optimal cell sphere balling pattern was tested by different methods.

The experimental process comprises the following steps: the cells in the culture flask were digested and collected by 0.125% of pancreatic enzyme, counted, and a sufficient amount of the cells were taken out according to the following table, respectively, and cell pellets were prepared according to different methods, respectively. Low adsorption plate: after resuspension with the appropriate amount of medium, the cell suspension was added to a low adsorption plate. A suspension drop method: after resuspension with the appropriate amount of medium, the cell suspension was dropped onto the dish lid using a line gun and inverted to suspend the drop onto the dish lid. 3D support: the cells were centrifuged and resuspended in 1ml of culture medium, then slowly and evenly dropped onto the 3D scaffold, placed in an incubator and allowed to stand for 30 minutes, after which the appropriate amount of culture medium was added to the container of the 3D scaffold. 3D microcarrier: the 3D microcarriers are infiltrated with a small amount of culture medium, after which the cell suspension is added to the container containing the 3D microcarriers. 2D culture: the cell suspension was inoculated into a culture flask.

The sample was tested for the proliferation inhibitory ability of immune cells according to the method of example 2 using the above sample. An appropriate amount of the sample was taken out from the above sample and tested for the content of PGE-2 according to the instructions of the ELISA kit.

Table 6 shows the different cell balling patterns. The change of paracrine capacity of cells in different culture systems is defined by taking the inhibiting capacity of immune cells as an evaluation standard.

TABLE 6

The content of PGE-2 in the culture supernatant was measured by ELISA. The results are shown in Table 7.

TABLE 7

FIG. 6 shows the amount of secreted PGE-2 in different culture protocols. Table 8 shows the proliferation inhibitory potency of the immune cells in different culture modes. FIG. 7 shows the proliferation inhibitory activity of the culture supernatants on immune cells.

TABLE 8

And (4) conclusion: compared with different balling modes, the balling mode with the bracket is closer to cells cultured in 2D in function, and the balling mode is superior to the balling mode with the bracket in function and shape, and the balling method with the bracket is more uniform in the balling method with the suspension drop method. The content measurement of the inflammation inhibitory factor PGE-2 in the culture supernatant proves that the PGE-2 content secretion amount of the cell balls cultured by the suspension drop method is the highest in a plurality of different cell culture modes, and the cell balls prepared by the suspension drop method are the best in function after being co-cultured with NK cells.

Example 5 Effect of cell sphere size on cell sphere diameter and function

The size of MSCs spheres affects cell function and viability, so that the size of spheres formed from different cell numbers is assessed and the change in size of cells in ringer's fluid is measured.

The experimental process comprises the following steps: corresponding MSCs cells were taken out according to the set of groups, resuspended in a culture medium to a cell suspension, and the cell pellet with different cell numbers was prepared according to the preparation method of the pendant drop method in example 2. After culturing for 48 hours in a 37 ℃ incubator containing 5% CO2, an appropriate amount of the cell pellet was taken out and the diameter of the cell pellet was measured with a scale under a microscope. Then replacing the culture medium with ringer's solution, and incubating the cells in an incubator at 1-10 deg.C. An appropriate amount of cell balls were taken out every day and observed under a microscope. Four groups A, B, C and D were set for comparison, as shown in the design scheme of Table 9, and FIG. 8 shows the cell morphology and size after sphering with different cell numbers. Table 10 shows the diameter of the spheronized cells.

TABLE 9

Group of	Number of cells per sphere	Time of balling
			A	0.5*10^4	48h
B
		1*10^4	48h
C
		2*10^4	48h
D
		3*10^4	48h

Watch 10

And (4) conclusion: the shapes and sizes of cells are different after different cell numbers are formed into spheres, when the cell spheres formed by the larger or smaller cell numbers are easy to be irregular, the function of the undersized cell spheres is poor, and the oversized cell spheres are easy to form necrotic centers to be unfavorable for cell survival.

Example 6 State before cell spheronization optimum test for spheronization variability and cell spheronization time

1. Difference of state before cell balling

The activity of the cells before balling has certain influence on the balling property of the cells, and the balling property of the recovered cells and the passaged cells is tested to determine which balling property is better.

The experimental process comprises the following steps: freezing cell ball samples: taking a frozen MSCs cell, quickly re-dissolving in a water bath, then adding into 10ml of physiological saline, centrifugally cleaning the cell, then carrying out constant volume counting, taking out a proper amount of cells, and preparing a cell ball according to the scheme of the pendant drop method.

Balling of cells in culture: the cells in the flask were digested with 0.125% pancreatin, the digestion was stopped by the culture supernatant, and the cells were washed and counted. Appropriate amount of cells were taken out and cell pellets were prepared according to the protocol of the above hanging drop method.

Table 11 shows the cell spheronization protocol and table 12 shows the results.

TABLE 11

TABLE 12

Fig. 9 shows the passaged cell sphering state and the revived cell sphering state, conclusion: the cell used for direct recovery has poor activity, is not easy to form a single sphere in the process of balling, and the balling of the passaged cells can stably form an independent cell sphere in the liquid drop, thereby being beneficial to stabilizing the quality of the cell sphere.

2. Cell balling time optimization test

Since cells need a certain time to form cell spheres, but the volume of the culture medium of the hanging drop is small, which is not beneficial to the survival of the cells, the optimal cell sphere forming time is found by testing the cell sphere forming time.

The experimental process comprises the following steps: digesting the cultured cells with 0.125% trypsin, taking out an appropriate amount of the cells, preparing into cell spheres according to the above-mentioned hanging drop method, and placing the cell spheres in 5% CO ₂ The cultivation was carried out in the incubator according to the following table.

Table 13 shows the cell spheronization time protocol experiments and cell spheronization status. The different grouping of balling states is shown in fig. 7.

Watch 13

The results in FIG. 10 show that cells do not form cell spheres efficiently within 24h, while 72h cell spheres lead to the generation of necrotic centers due to the lack of nutrients, and 48h is the optimal spheronization time.

EXAMPLE 7 optimal test for spheroblast droplets and whether incubation was preceded by low adsorption plate culture

1. Optimal test for spheroblast droplets

Cells need a culture medium as a resuspension in a suspension drop balling culture process, and the optimal drop volume is confirmed by monitoring balling.

The experimental process comprises the following steps: the cells in culture were digested with 0.125% trypsin, and the appropriate amount of cells was taken out to resuspend the cells in the volume of the medium in the following table, after which they were prepared into cell pellets according to the method of the above-mentioned pendant drop method. Culturing was carried out for 48h in an incubator containing 5% CO2, and the state of the cell pellet was observed and evaluated after 48 h. Table 14 shows the effect of different droplets on cell balling.

TABLE 14

And (4) conclusion: the cell drops are prepared by a pendant drop method, the maximum suspendable liquid drop is 40ul, excessively large or small liquid drops are not beneficial to cell balling, and 30ul liquid drops are the most suitable liquid drop amount.

2. Whether the culture is carried out by a low adsorption plate before hatching

The state of the cell pellet can be further promoted by a short culture of the cell pellet as a culture medium before transferring into ringer's solution, and thus whether the cell pellet is cultured or not is tested.

The experimental process comprises the following steps: digesting the cultured cells with 0.125% trypsin, taking out an appropriate amount of cells, preparing the cells into spheroids according to the method of the pendant drop method in the embodiment 2, culturing the spheroids in an incubator with 5% CO2 for 48h, collecting the spheroids of the group A into a culture plate, adding an appropriate amount of culture medium, culturing the spheroids in the incubator for 48h, culturing the spheroids of the group B in a refrigerator with 1-10 ℃ by using physiological saline for 48h, and observing the states of the spheroids of the two groups, wherein the cell numbers of the groups A and B are 2 x 10^4 cells/sphere.

FIG. 11 shows the state of the group A cell spheres with smoother cell sphere edges; the cell ball edge is rough in the state of the B cell ball of the group. And (4) conclusion: the cell balls have no obvious change in diameter, but the cell balls cultured by the low adsorption plate have smoother and more compact cell balls, so that the cell balls are more favorable for cell survival.

EXAMPLE 8 incubation temperature and Effect of liquid on cell pellets

1. Effect of incubation temperature on cell pellets

Incubation is required to collect cell secretion capacity after cell pellet formation, and the optimal temperature and cell hatching fluid are tested with cell survival and death as test targets in order to ensure optimal incubation temperature.

The experimental process comprises the following steps: the cultured cells were digested with 0.125% trypsin, an appropriate amount of the cells were taken out to prepare cell pellets according to the hanging drop method, the cells were cultured in an incubator 5% CO2 for 48 hours, then the cell pellets were collected, the cell pellets were washed with physiological saline to remove the residual medium, the cells were grouped according to the following Table 15, incubated for 48 hours under different incubation liquids and different temperature conditions, and then an appropriate amount of the cell pellets were taken out to stain the survival/death state of the cells with DAPI/PI, and observed under a microscope. Table 16 shows the results of the testing of the incubation temperature and incubation fluid for each group of pellets, and fig. 12 shows the effect of the incubation temperature on the survival status of the pellets.

Watch 15

TABLE 16

And (4) conclusion: the incubation of the cell balls by designing different temperatures shows that the cell balls can keep better activity in a group at 1-10 ℃ and a high-temperature group, and only saline is used as an incubation liquid to ensure that the cells can still keep the survival ability in the low temperature.

2. Hatching fluid effects on cell pellets

The cell balls can be preserved in saline water and play a proper role, and in order to select the cell ball hatching fluid with the best preservation effect, a conventional hatching fluid experiment which uses more reinfusion fluid clinically as cells is selected.

The experimental process comprises the following steps: the cultured cells were digested with 0.125% trypsin, an appropriate amount of the cells were taken out and prepared into cell balls according to the above-described hanging drop method, cultured in an incubator 5% CO2 for 48 hours, and then the cell balls were collected, the cell balls were washed with physiological saline to remove the residual medium, grouped according to the following Table 17, incubated in different incubation liquids for 7 days, and then an appropriate amount of the cell balls were taken out and stained for the survival/death state of the cells with DAPI/PI, and observed under a microscope.

TABLE 17

FIG. 13 shows the state of the A-C group cell balls observed under a fluorescence microscope, in conclusion: after 7 days of incubation, the cell survival rate of the cells in the ringer and the glucose injection is higher, but after subsequent tests, the glucose injection as the incubation liquid is not favorable for the survival of immune cells. EXAMPLE 9 Effect of different injections on the survival of immune cells and incubation time test

1. Effect of different hatching injections on the survival of immune cells

The cell protective solution prepared by different hatching solutions has different influences on the protective capability of the cells, so the prepared protective solution and the cells are hatched for 24 hours, and the influences of the different hatching solutions on the cells are observed.

The experimental process comprises the following steps: washing an appropriate amount of high-activity immune cells twice by using normal saline to remove residual culture medium, taking an appropriate amount of prepared cell protection solution as cell resuspension solution to be mixed and resuspended with the immune cells, placing the cell protection solution in an environment with the temperature of 1-10 ℃ for 24h, mixing uniformly after 24h, taking 10ul of cell suspension solution to be mixed with 10ul of trypan blue staining solution for staining, and calculating the cell viability by using a cell counter.

The effect on immune cell activity was investigated using different hatching fluid species, respectively, and the results are shown in fig. 14. And (4) conclusion: the protection effect of different hatching solutions on cells is different, the glucose injection can better maintain the state of cells in a cell ball, and the glucose injection has an inhibiting function when being co-cultured with immune cells, but the cell survival rate of the immune cells is obviously reduced, which indicates that the glucose hatching solution does not play a role in protecting the immune cells.

2. Incubation time test

Tests were performed to verify the time that the cell pellet could survive under optimal conditions.

The experimental process comprises the following steps: digesting the cultured cells with 0.125% trypsin, taking out an appropriate amount of the cells, preparing into cytospheres according to the method of the pendant drop method of example 2, culturing in an incubator of 5% CO2 for 48h, then collecting the cytospheres, washing the cytospheres with physiological saline to remove the residual medium, grouping according to the following Table 17, incubating for various times in ringer's solution, then taking out an appropriate amount of the cytospheres, staining the survival/death status of the cells with DAPI/PI, and observing under a microscope.

Watch 18

FIG. 15 shows the cell pellet morphology of groups A-D. And (4) conclusion: the cytospheres can keep the cell viability for a long time in the long-time hatching process until the cytospheres are proved to have high activity by live cell staining before the group C, and the cells can be stained by a dead cell staining agent PI at the time point of the group D, which indicates that the cytospheres are in a cell survival state before the group D.

Example 10 experiment of cell diameter variation

The size of MSCs spheres affects the function and viability of cells, so the size of spheres formed from different cell numbers was evaluated and the size change of cells in ringer's solution was measured.

The experimental process comprises the following steps: and respectively taking out corresponding MSCs according to the group settings, re-suspending the MSCs into cell suspension by using a culture medium, and preparing cell balls with different cell numbers according to the preparation method of the hanging drop method. After culturing for 48h in a 37 ℃ incubator containing 5% CO2, an appropriate amount of the cell pellet was taken out and the diameter of the cell pellet was measured with a ruler under a microscope. Then replacing the culture medium with ringer's solution, and incubating the cells in an incubator at 1-10 deg.C. An appropriate amount of the cell pellet was taken out every day and observed and measured under a microscope.

Watch 19

The change in cell sphere diameter is shown in FIG. 16. And (4) conclusion: the cell spheres varied in diameter after formation of spheres at different cell numbers, but the size of the cell spheres varied less over time. Indicating that the cell balls remained relatively stable in morphology during the long-term incubation.

EXAMPLE 11 post-hatch identification of cell pellets

And (3) after the cell balls are cultured, identifying the cells of the cell balls in order to identify whether the cells in the cell balls are MSCs.

The experimental process comprises the following steps: the cell pellet was prepared according to the cell pellet preparation method, incubated at 1-10 ℃ for 7 days, then taken out, divided into 3 parts, the first part was added with the MSC culture medium, transferred into a culture flask, and placed in a 37 ℃ incubator containing 5% CO2 for culture. Cells in the cell pellet were observed after 3 days for adherence. The second cell pellet was digested with 0.125% pancreatic enzyme, the digestion was terminated with medium, the digested cells were washed with physiological saline, and then cell surface markers were detected by flow cytometry. Digesting the third cell ball with 0.125% pancreatin, terminating the cell ball with a culture medium after digestion, cleaning the cell with normal saline, respectively inoculating the cell into a adipogenic differentiation culture medium, an osteogenic differentiation culture medium and a chondrogenic differentiation culture medium, carrying out differentiation culture on the MSC according to the specification of the differentiation kit, and finally identifying the differentiation result by using a staining agent in the differentiation kit. Table 20 shows the cell identification method.

Watch 20

Fig. 17 shows the results of the cell balls in the normal culture flask, fig. 18 shows the phenotypic flow assay of MSCs, and fig. 19 shows the results of the adipogenic differentiation assay, the chondrogenic differentiation assay, and the osteogenic differentiation assay, respectively. And (4) conclusion: the cell balls still have the characteristics of the MSCs after 3D culture, which indicates that the MSCs cells are not differentiated in the 3D culture process.

EXAMPLE 12 test of protective Effect of cell pellet suspension

The effect of the cell protective solution on cell preservation was tested.

The experimental process comprises the following steps: preparing a cell ball suspension, taking out a proper amount of the frozen and preserved MSC cells, quickly redissolving the cells in a water bath, adding the cells into 10ml of physiological saline, washing twice, counting the cells by using a cell counter, taking out the MSC cells required in the following table 21, dividing the MSC cells into 4 groups, respectively using 1ml of ringer's solution and the cell ball suspension as resuspension, then preserving the cells in a refrigerator at 1-10 ℃, mixing 10ul of samples taken from the samples at intervals with 10ul of trypan blue staining solution, and calculating the survival rate and the cell number of the cells preserved at low concentration and high concentration by using the cell counter.

Table 21 shows the experiments on the survival rate of MSCs by cell pellet suspension, table 22 shows the experiments on the protection of immune cells by cell pellet suspension, and table 23 shows the experiments on the preservation of high concentration cells.

TABLE 21

TABLE 22

TABLE 23

The cell can still provide 79% of survival rate for 53 hours under the conventional cell concentration.

Watch 24

Table 24 shows the results of the protection experiments on MSCs by suspension, and fig. 20 shows a count chart of MSCs in suspension.

The cell ball suspension also has a certain protection effect on immune cells. Table 25 shows the results of the protective effect of the suspensions on immune cells, and Table 26 shows the protective effect of the suspensions for high concentration cell resuspension

TABLE 25

Watch 26

And (4) conclusion: the suspension has prolonged cell viability retention time in the resuspension solution for both MSCs and immune cells, and high concentration cell preservation experiments result in 79% cell viability at 18h at 4 x 10A 7, and greater than 80% cell viability at 24h at 2 x 10A 7, whereas the cell density is 1-2 x 10A 6/mL for conventional clinical use. The product has a better application range.

Example 13 test of inflammation inhibitory Effect of cell suspension

The product also has an immunosuppressive effect, and the function of the product is tested by the proliferation inhibitory effect on NK. Table 27 is the experimental design for suppression of proliferation of immune cells by suspension.

Watch 27

FIG. 21 shows the inhibition of immune cell proliferation assay by suspension. And (4) conclusion: the suspension has a good cell preservation effect, a good proliferation inhibition effect on immune cells and a good effect on diseases caused by chronic inflammation such as arthritis.

EXAMPLE 14 cell ball suspension toxicity test

To test whether the MSC pellet suspension is toxic to cells under this protocol, the protective solution was tested for toxicity to human hepatocytes by CCK-8.

The experimental process comprises the following steps: reviving human liver cells, culturing with liver cell culture medium until liver cells mature, and preparing into MSC globulin suspension. Removing the culture medium in the hepatocyte culture hole, adding fresh hepatocyte culture medium, adding cell suspension in equal proportion into the sample hole, adding ringer's solution into the negative control hole, and adding cyclosporin A with toxic effect on hepatocyte into the positive hole except for adding ringer's solution in equal proportion. After incubation in the incubator for 24h, each well was supplemented with CCK-8, which was then returned to the incubator for further incubation for 2h. And taking out the cell plate, testing the absorbance by using an enzyme-labeling instrument, and calculating the activity of the liver cells. The experimental design is shown in table 28 below. The results are shown in FIG. 22, which indicates that the cell pellet suspension is not toxic to hepatocytes and immune cells.

Watch 28

Example 15

The mesenchymal stem cells are recovered, inoculated according to the density of 5000-7000 cells/cm 2, cultured in an incubator with 5 percent of CO2 at 37 ℃, digested until the cells grow to 70-80 percent, the pancreatin use concentration is 0.125 percent, the digestion time is 3 minutes, the digested cells are washed for 2 times, and then re-suspended by using an MSC culture medium, and the re-suspended concentration is 1w of cells/100 ul. Dropping the cell resuspension solution on an inner surface cover of a 60mm plate by using a 100-ul discharging gun after resuspension, adding a small amount of DPBS into the plate, quickly turning over the cover containing the cell resuspension solution to cover, slowly putting the plate into an incubator to be cultured, wherein the incubator conditions are 37 ℃ and 5% CO2, transferring the cell balls into a low adsorption plate after the cells are cultured for 2 days, adding a proper amount of culture medium into the low adsorption plate to be cultured for one day, collecting the cell balls after culturing, washing the cell balls for 2 times by using pbs, then carrying out resuspension by using physiological saline, putting the cell balls into a refrigerator with the temperature of 1-10 ℃ for incubation, wherein the concentration of the resuspension solution is 300 cell balls/50 ml, and placing the cell balls in the refrigerator for 4-9 days. Filtering the cultured physiological saline by using a 0.22um filter screen, using the filtered physiological saline as the final heavy suspension of cells after the fibroblast is recovered or is subjected to subculture digestion, preparing an injection according to the concentration of 1-2 x 10^7 cells/ml, and putting the injection into an incubator at 0-15 ℃ for long-distance transportation and direct use, wherein the preparation process is shown in figure 1.

Example 16

The mesenchymal stem cells were recovered, inoculated at a normal density, cultured in an incubator containing 5% of CO2 at 37 ℃, digested to the extent of 70 to 80% of the cells grown, and the use concentration of pancreatin was 0.125%, the digestion time was 3 minutes, and the digested cells were washed 2 times, and then resuspended in a DMEM medium supplemented with 5% of pancreatin at a resuspension concentration of 1w cells/100 ul. Dropping the cell resuspension solution on an inner surface cover of a 60mm plate by using a 100-ul discharging gun after resuspension, adding a small amount of DPBS into the plate, quickly turning over the cover containing the cell resuspension solution to cover, slowly putting the plate into an incubator to be cultured, wherein the incubator conditions are 37 ℃ and 5% CO2, transferring the cell balls into a low adsorption plate after the cells are cultured for 2 days, adding a proper amount of culture medium into the low adsorption plate to be cultured for one day, collecting the cell balls after culturing, washing the cell balls for 2 times by using pbs, then carrying out resuspension by using physiological saline, putting the cell balls into a refrigerator with the temperature of 1-10 ℃ for incubation, wherein the concentration of the resuspension solution is 300 cell balls/50 ml, and placing the cell balls in the refrigerator for 4-9 days. Filtering the cultured physiological saline by using a 0.22um filter screen, using the filtered physiological saline as a final heavy suspension of cells after the fibroblast is recovered or is subjected to subculture digestion, preparing the physiological saline according to the concentration of 5 x 10^7 cells/100 ml, transferring the physiological saline into a transfer bag, and putting the transfer bag into an incubator at the temperature of 0-15 ℃ for long-distance transportation and direct use.

Example 17

The frozen mesenchymal stem cells are recovered, inoculated according to the density of 5000-7000 cells/cm 2, cultured in an incubator with 5 percent CO2 at 37 ℃, digested until the cells grow to 70-80 percent, the pancreatin use concentration is 0.125 percent, the digestion time is 3 minutes, the digested cells are washed for 2 times, and then resuspended in MSCs culture medium with the resuspension concentration of 1w of cells/100 ul. Dropping the cell resuspension solution on an inner cover of a 60mm plate by using a 100-ul discharging gun after resuspension, adding a small amount of DPBS into the culture dish, quickly turning over the cover containing the cell resuspension solution to cover, slowly putting the culture dish into an incubator to be cultured, wherein the incubator is 5 percent of CO2 at 37 ℃, culturing for 2 days until cells form a cell ball, transferring the cell ball into a low adsorption plate, adding an appropriate amount of culture medium into the incubator to be cultured for one day, collecting the cell ball after culturing, washing for 2 times by using pbs, then carrying out resuspension by using ringer's solution, putting the cell ball into a refrigerator at 1-10 ℃ to be incubated, wherein the concentration of the resuspension solution is 300 cell balls/50 ml, and placing the cell ball in the refrigerator for 4-9 days. Filtering the cultured ringer's solution with a 0.22um filter screen, using the filtered ringer's solution as the final heavy suspension of cells after the recovery or passage digestion of fibroblasts, preparing into injection according to the concentration of 1-2 x 10^7 cells/ml, and putting into an incubator at 0-15 ℃ for long-distance transportation and direct use.

Example 18

The mesenchymal stem cells are recovered, inoculated according to the density of 5000-7000 cells/cm 2, cultured in an incubator with 5 percent of CO2 at 37 ℃, digested until the cells grow to 70-80 percent, the pancreatin use concentration is 0.125 percent, the digestion time is 3 minutes, the digested cells are washed for 2 times, and then resuspended by MSCs culture medium with the resuspension concentration of 1w of cells/100 ul. Dropping the cell resuspension solution on an inner surface cover of a 60mm plate by using a 100-ul discharging gun after resuspension, adding a small amount of DPBS into the plate, quickly turning over the cover containing the cell resuspension solution to cover, slowly putting the plate into an incubator to be cultured, wherein the incubator conditions are 37 ℃ and 5% CO2, transferring the cell balls into a low adsorption plate after the cells are cultured for 2 days, adding a proper amount of culture medium into the low adsorption plate to be cultured for 1 day, collecting the cell balls after culturing, washing the cell balls for 2 times by using pbs, then carrying out resuspension by using ringer's solution, incubating at 10-30 ℃, wherein the concentration of the resuspension solution is 300 cell balls/50 ml, and standing for 4-9 days. Filtering the cultured ringer's solution by using a 0.22um filter screen, using the filtered ringer's solution as the final heavy suspension of cells after the recovery or passage digestion of mesenchymal stem cells, preparing into injection according to the concentration of 1-2 x 10^7 cells/ml, and putting into an incubator at 0-15 ℃ for long-distance transportation and direct use.

Example 19

The mesenchymal stem cells are recovered, inoculated according to the density of 5000-7000 cells/cm 2, cultured in an incubator with 5 percent of CO2 at 37 ℃, digested until the cells grow to 70-80 percent, the pancreatin use concentration is 0.125 percent, the digestion time is 3 minutes, the digested cells are washed for 2 times, and then resuspended by MSCs culture medium with the resuspension concentration of 1w of cells/100 ul. Dropping the cell resuspension solution on an inner cover of a 60mm plate by using a 100-ul discharging gun after resuspension, adding a small amount of DPBS into the plate, quickly turning over the cover containing the cell resuspension solution, slowly putting the plate into an incubator for culturing, wherein the incubator is 5 percent of CO2 at 37 ℃, after the cells are cultured for 2 days, transferring the cell spheres into a low adsorption plate after the cells form the cell spheres, adding an appropriate amount of culture medium into the incubator for culturing for one day, collecting the cell spheres after culturing, washing the cell spheres for 2 times by using pbs, then carrying out resuspension by using physiological saline, putting the cell spheres into a 0 ℃ refrigerator for incubation, wherein the concentration of the resuspension solution is 300 cell spheres/50 ml, and putting the cell spheres in the refrigerator for 4-9 days. Filtering the cultured normal saline by using a 0.22um filter screen, recovering or subculturing the filtered normal saline as the final heavy suspension of the cells, preparing the final heavy suspension into injection according to the concentration of 1-2 x 10^7 cells/ml, and putting the injection into an incubator at the temperature of 0-15 ℃ for long-distance transportation and direct use.

Example 20

FIG. 23 shows the morphology of the spheroids of 1w cells prepared by the method of the present invention transferred to a low adsorption plate after two days of culture, and FIG. 24 shows that the cells of 1w cells prepared by the method of the present invention survive within the spheroids after 7 days of incubation. FIG. 25 shows that the cell balls prepared by the method of the present invention can still have adherence performance when placed in a wall plate.

Table 29 shows the cell viability rate and cell number of the suspensions of physiological saline and fibroblast cells as resuscitated cells over time.

TABLE 29

Table 30 shows the cell viability and cell number of the suspension of ringer's solution and MSCs cell pellets as resuscitated cells over time.

Watch 30

FIG. 26 shows the state of the cells after 20h after staining of MSCs with Trypan blue.

In the description herein, reference to the description of the terms "one embodiment," "some embodiments," "an example," "a specific example," "some embodiments," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that changes, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims (8)

1. Use of a cell preservation solution for the preparation of an immunosuppressant, wherein the cell preservation solution is prepared by the following method:

(1) Preparing a 3D cell ball;

(2) Resuspending and incubating the 3D cell balls to obtain a cell ball suspension;

(3) Removing the cell components from the cell pellet suspension to obtain a cell preservation solution,

wherein the incubation temperature is 0-37 ℃, and the incubation time is 2-170 h.

2. Use according to claim 1, characterized in that the cells contained in the 3D cell spheres are selected from mesenchymal cells;

optionally, the cells contained in the 3D cell spheres are selected from at least one of fibroblasts, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells.

3. The use according to claim 1, wherein the method of forming 3D cell spheres comprises self-sphere and carrier sphere.

4. The use of claim 1, wherein in the step (2), the resuspension solution used for resuspending the 3D cell pellet is at least one selected from the group consisting of sodium chloride injection, sodium chloride compound injection, lactated ringer's injection, and glucose injection.

5. Use according to claim 1, characterized in that the method for the preparation of 3D cell spheres comprises:

1) Taking out the frozen cells for redissolution and resuspension;

2) Culturing the resuspended cells in a DMEM medium containing 4-8% FBS;

3) Subjecting the cultured cells to trypsinization to obtain digested cells;

4) Resuspending the digested cells to obtain a cell resuspension solution;

5) And inoculating the cell resuspension into a culture dish, and culturing to obtain the 3D cell ball.

6. Use according to claim 5, characterized in that the cells contained in the 3D cell spheres are selected from mesenchymal cells;

optionally, the cells contained in the 3D cell spheres are selected from at least one of fibroblasts, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells.

7. The use according to claim 6, wherein in step 4) the digested cells are resuspended using physiological saline.

8. The use of claim 1, wherein the immunosuppressive agent comprises mesenchymal stem cells.

Images